HK1215792B - Bakuchiol compositions for treatment of post inflammatory hyperpigmentation - Google Patents

Description

Bakuchiol composition for treating post-inflammatory hyperpigmentation

Citation of Related Applications

This application claims the benefit under 35 U.S.C. §119(e) of PCT Application No. US2011/026594, filed on March 1, 2011, and U.S. Provisional Patent Application No. 61/438,890, filed on February 2, 2011.

background

Technical Field

The present invention generally relates to bakuchiol compositions and their use for treating post-inflammatory hyperpigmentation.

Related technical description

Post-inflammatory hyperpigmentation (PIH) is a rare skin pigmentation disease state associated with increased melanin synthesis and deposition. PIH is also characterized by apoptosis of melanocytes produced by invasion of mediators and cytokines from inflammatory and immune responses due to oxidative stress. Melanin deposition (i.e., excessive pigmentation) occurs beyond the epidermal layer with significant melanin release into the papillary dermis and is captured by large immune cells. These unique histological features of PIH present numerous difficulties for treating PIH using conventional pharmaceutical agents.

Common treatments for PIH focus on preventing further pigmentation by controlling inflammation with corticosteroids and photoprotectants. Chemical peels, such as salicylic acid and glycolic acid, are also used to stimulate skin renewal and remove or reduce pigmentation. Topical retinoids have also been used to treat PIH, but these approaches can take up to 40 weeks before significant benefits are seen.

Tyrosinase inhibitors or skin whitening agents such as hydroquinone, azelaic acid, kojic acid and liquorice extract have also been used to treat PIH. A significant disadvantage of using conventional skin whitening agents or tyrosinase inhibitors is the widespread fading of normal skin adjacent to the PIH site. This effect weakens the color of the background skin and makes the PIH site more prominent. Therefore, these agents must be applied very carefully at the PIH site. In addition, tyrosinase inhibitors are only effective for epidermal hyperpigmentation because the epidermis is the site where melanin is synthesized by tyrosinase. Since post-inflammatory pigmentation occurs in the deep layers of the skin (e.g., papillary dermis), the application of hydroquinone drugs for more than 6 consecutive months is required before visible changes in black marks are observed. Finally, hydroquinone type skin whitening agents or tyrosinase inhibitors are associated with side effects, including skin irritation, dryness, teratogenicity, and the induction of vitiligo and skin cancer.

Post-inflammatory hyperpigmentation can be caused by endogenous inflammatory skin diseases such as acne, atopic dermatitis, allergic contact dermatitis, incontinentia pigmenti, lichen planus, lupus erythematosus, and morphea. Other causes of PIH include exogenous inflammatory stimuli such as mechanical trauma, ionizing and non-ionizing radiation, burns, laser therapy, and skin infections. Current therapeutic agents for the above-mentioned skin conditions are ineffective for preventing, alleviating, reducing, or treating PIH. For example, anti-inflammatory agents such as tretinoin, COX inhibitors (e.g., salicylic acid), nonsteroidal anti-inflammatory drugs (NSAIDs), antibacterial agents, or hormone drugs are often used to treat the above-mentioned skin conditions, but these treatments have been shown to be ineffective for PIH.

Despite significant progress in this area, there remains a significant need for methods to prevent, alleviate, reduce, or treat hyperpigmentation. For example, there is a need for methods to treat post-inflammatory hyperpigmentation. The present invention addresses these needs and provides further related advantages.

Overview

In summary, the present invention relates to methods for preventing, alleviating, reducing, or treating hyperpigmentation. Hyperpigmentation can be the result of a disease state originating from an inflammatory skin disease state. For example, one embodiment of the present invention is a method for preventing, alleviating, reducing, or treating post-inflammatory hyperpigmentation (PIH). This PIH can result from a variety of skin conditions, including acne. The method comprises administering to a mammal an effective amount of a composition comprising bakuchiol and less than 500 ppm of total furanocoumarin impurities.

In contrast to other skin whitening agents, the bakuchiol compositions disclosed herein are not tyrosinase inhibitors. Thus, the disclosed compositions specifically depigment at the site of PIH and are useful for treating hyperpigmentation in deeper layers of the skin (e.g., the papillary dermis). Thus, the presently disclosed methods include several advantages over previous methods for treating hyperpigmentation and/or PIH.

Thus, one embodiment of the present disclosure is directed to a method for preventing, alleviating, reducing or treating hyperpigmentation caused by a condition resulting from an inflammatory skin disorder, comprising administering to a mammal an effective amount of a composition comprising bakuchiol or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and less than 500 ppm of total furanocoumarin impurities.

In some embodiments, the disease state is post-inflammatory hyperpigmentation. In other embodiments, the composition comprises less than 100 ppm of total furanocoumarin impurities. In other embodiments, the furanocoumarin impurities comprise psoralen, isopsoralen, or a combination thereof. In some embodiments, the composition does not exhibit tyrosinase inhibitory activity relative to a kojic acid control.

In other embodiments, bakuchiol is chemically synthesized or isolated from a plant. For example, in some embodiments, bakuchiol is isolated from a plant. In other embodiments, the plant is from the genus Psoralea, such as Psoralea corylifolia L. (Leguminosae) or Psoralea aglandulosa L. (Fabaceae).

In other embodiments, bakuchiol is isolated from seeds, stems, bark, twigs, tubers, roots, root bark, sprouts, rhizomes, flowers or other reproductive organs, leaves or other aerial parts, or combinations thereof.

In some other embodiments, post-inflammatory hyperpigmentation (PIH) is caused by acne, atopic dermatitis, allergic contact dermatitis, incontinentia pigmenti, lichen planus, lupus erythematosus, morphea, mechanical trauma, ionizing or non-ionizing radiation, burns, laser or drug treatment, skin infection, or a combination thereof. For example, in some aspects, post-inflammatory hyperpigmentation (PIH) is caused by acne.

In other embodiments, the composition comprises 0.001% to 99.9% total weight of bakuchiol and a pharmaceutically, dermatologically, or cosmetically acceptable carrier. For example, in some aspects, the composition comprises 0.1% to 2.0% total weight of bakuchiol, 1.0% total weight of bakuchiol, or 0.5% total weight of bakuchiol.

In other embodiments, the dermatologically acceptable carrier comprises a non-stick gauze, bandage, cotton swab, wipe, plaster, mask or protectant. In some other embodiments, the cosmetically acceptable carrier comprises a cleanser or antibacterial agent.

In some aspects, the composition is formulated for topical administration. For example, in some aspects, the composition further comprises a cream, a lotion, an ointment, a gel, an emulsion, a liquid, a paste, a soap, a powder, or a combination thereof.

In other embodiments, the composition further comprises an adjuvant, a skin penetration enhancer, or a liposome. In other embodiments, the adjuvant comprises an α-hydroxy acid, salicylic acid, linoleic acid, tretinoin, benzoyl peroxide, sodium sulfacetyl, clindamycin, erythromycin, dapsone, tetracycline, deoxycycline, minocycline, zinc, estrogen or its derivatives, antiandrogens, sulfur, steroids, cortisone, tazarotene, curcumin extract, gum arabic extract, scutellaria baicalensis extract, green tea extract, grape seed extract, or a combination thereof.

In some embodiments, the composition is formulated in capsule form, e.g., a controlled release capsule. In other embodiments, the composition is administered topically by aerosol, by suppository, intradermally, intramuscularly, or intravenously.

In some aspects, the methods prevent hyperpigmentation. In other aspects, the methods alleviate hyperpigmentation. In other aspects, the methods reduce hyperpigmentation. In other aspects, the methods treat hyperpigmentation.

In other embodiments, the hyperpigmentation occurs in the deeper layers of the skin, for example, in the papillary dermis. In other embodiments, the method further comprises reducing superoxide anions. In some other embodiments, the method further comprises reducing melanogen production. In other embodiments, the method further comprises reducing melanocyte proliferation. In other embodiments, the method further comprises preventing melanocyte apoptosis.

In some other embodiments, the mammal is a human. In some other embodiments, the mammal is in need of preventing, alleviating, reducing, or treating hyperpigmentation caused by a condition resulting from an inflammatory skin disorder. For example, the mammal may be in need of treatment for PIH.

In another embodiment, the present disclosure relates to a method for reducing melanogenesis, reducing melanocyte proliferation, or preventing melanocyte apoptosis, comprising administering to a mammal an effective amount of a composition comprising bakuchiol or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier and less than 500 ppm of total furanocoumarin impurities. In some other embodiments, the method further comprises reducing superoxide anions.

In other embodiments, the composition comprises less than 100 ppm of total furanocoumarin impurities. In other embodiments, the furanocoumarin impurities comprise psoralen, isopsoralen, or a combination thereof. In some embodiments, the composition exhibits no tyrosinase inhibitory activity relative to a kojic acid control.

In other embodiments, bakuchiol is chemically synthesized or isolated from a plant. For example, in some embodiments, bakuchiol is isolated from a plant. In some other embodiments, the plant is from the genus Psoralea, such as Psoralea corylifolia L. (Fabaceae) or Psoralea aglandulosa L. (Fabaceae).

In other embodiments, bakuchiol is isolated from seeds, stems, bark, twigs, tubers, roots, root bark, sprouts, rhizomes, flowers or other reproductive organs, leaves or other aerial parts, or combinations thereof.

In some embodiments, melanogenesis, melanocyte proliferation, or melanocyte apoptosis is the result of post-inflammatory hyperpigmentation (PIH). In some other embodiments, post-inflammatory hyperpigmentation (PIH) is caused by acne, atopic dermatitis, allergic contact dermatitis, incontinentia pigmenti, lichen planus, lupus erythematosus, morphea, mechanical trauma, ionizing or non-ionizing radiation, burns, laser or drug treatment, skin infection, or a combination thereof. For example, in some aspects, post-inflammatory hyperpigmentation (PIH) is caused by acne.

In other embodiments, the composition comprises 0.001% to 99.9% total weight of bakuchiol and a pharmaceutically, dermatologically, or cosmetically acceptable carrier. For example, in some aspects, the composition comprises 0.1% to 2.0% total weight of bakuchiol, 1.0% total weight of bakuchiol, or 0.5% total weight of bakuchiol.

In other embodiments, the dermatologically acceptable carrier comprises a non-stick gauze, bandage, cotton swab, wipe, plaster, mask or protectant. In some other embodiments, the cosmetically acceptable carrier comprises a cleanser or antibacterial agent.

In some aspects, the composition is formulated for topical administration. For example, in some aspects, the composition further comprises a cream, a lotion, an ointment, a gel, an emulsion, a liquid, a paste, a soap, a powder, or a combination thereof.

In other embodiments, the composition further comprises an adjuvant, a skin penetration enhancer, or a liposome. In other embodiments, the adjuvant comprises an α-hydroxy acid, salicylic acid, linoleic acid, tretinoin, benzoyl peroxide, sodium sulfacetyl, clindamycin, erythromycin, dapsone, tetracycline, deoxycycline, minocycline, zinc, estrogen or its derivatives, antiandrogens, sulfur, steroids, cortisone, tazarotene, curcumin extract, gum arabic extract, scutellaria baicalensis extract, green tea extract, grape seed extract, or a combination thereof.

In some embodiments, the composition is formulated in capsule form, e.g., a controlled release capsule. In other embodiments, the composition is administered topically by aerosol, by suppository, intradermally, intramuscularly, or intravenously.

In some aspects, the methods prevent hyperpigmentation. In other aspects, the methods alleviate hyperpigmentation. In other aspects, the methods reduce hyperpigmentation. In other aspects, the methods treat hyperpigmentation. In some embodiments, hyperpigmentation occurs in the deeper layers of the skin, for example, in the papillary dermis layer of the skin.

In some other embodiments, the methods reduce melanogenesis. In other embodiments, the methods reduce melanocyte proliferation. In other embodiments, the methods prevent melanocyte apoptosis.

In some other embodiments, the mammal is a human. In some other embodiments, the mammal is in need of treatment to reduce melanogenesis, reduce melanocyte proliferation, or prevent melanocyte apoptosis.

In other embodiments, the composition further comprises salicylic acid or a pharmaceutically acceptable salt thereof.

Other embodiments of the present disclosure relate to methods for treating inflammatory or non-inflammatory lesions, comprising administering to a mammal an effective amount of a composition comprising bakuchiol or a pharmaceutically acceptable salt thereof and salicylic acid or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. For example, in some embodiments, the lesions comprise inflammatory acne lesions. In other embodiments, the methods treat both inflammatory and non-inflammatory lesions.

In some other embodiments of the foregoing, the mammal is a human. In some other embodiments, the mammal is in need of treatment for an inflammatory or non-inflammatory condition.

In other embodiments, the present invention includes a composition comprising bakuchiol or a pharmaceutically acceptable salt thereof and salicylic acid or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. In some embodiments, the composition is formulated for topical administration.

These and other aspects of the invention will be apparent upon reference to the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings, like reference numbers represent similar elements. The sizes and relative positions of elements in the drawings are not necessarily drawn to scale, and some of these elements are arbitrarily enlarged and positioned to improve drawing legibility. Furthermore, the particular shapes of the elements as drawn are not intended to convey any information about the actual shape of the particular elements and are merely selected for ease of identification in the drawings.

Figure 1 depicts the chromatograms of bakuchiol, psoralen, and isopsoralen standards.

FIG2 shows chromatograms of bakuchiol compositions before and after hydrolysis.

FIG3 provides data showing the strong antioxidant properties of bakuchiol compositions.

FIG4 is a graph showing the tyrosinase inhibitory activity of bakuchiol compositions and kojic acid.

FIG5 shows the changes in PIH severity among the individual test subjects.

FIG6 is a graph showing the percent change in facial area affected by PIH for individual test subjects.

FIG7 shows PIH and the mean percent change in PIH severity for the five subjects tested.

Figure 8 depicts the reduction in mean grade level of PIH and PIH severity at each visit compared to baseline.

Figure 9 shows photos of two study participants at different time intervals.

Detailed description

In the following description, certain specific details are set forth to provide a thorough understanding of various embodiments. However, one skilled in the art will appreciate that the present invention can be practiced without these details. In other instances, well-known structures are not shown or described in detail to avoid unnecessarily obscuring the description of the embodiments. Unless the context requires otherwise, in the following description and claims, the word "comprise" and variations such as "comprises" and "comprising" are to be interpreted as having an open, inclusive meaning, i.e., for example, "including but not limited to." In addition, the headings provided herein are for convenience only and do not indicate the scope or meaning of the claimed invention.

In this specification, reference to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described with respect to the embodiment is included in at least one embodiment. Thus, the appearance of the phrase "in one embodiment" or "in an embodiment" in various places in this specification are not necessarily with respect to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. Furthermore, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that, in general, use of the term "or" includes the meaning of "and/or" unless the context clearly dictates otherwise.

definition

As used herein, and unless the context dictates otherwise, the following terms have the meanings as defined below.

As used herein, "bakuchiol" refers to a compound having the formula:

The benzyl double bond can be cis or trans. As used herein, bakuchiol includes pharmaceutically acceptable salts and tautomers of bakuchiol. Phenolic compounds structurally related to bakuchiol are also included in this definition.

"Bakutrol ^™ " is a composition comprising bakuchiol and may additionally comprise fatty acids extracted from the Psoralea plant.

"UP256" refers to a 0.5% (wt/wt) formulation of bakuchiol.

"Preventing," "prevention," and "prevent" in the context of the disclosed methods all refer to preventative methods that hinder or stop the occurrence of a particular disease state, such as PIH.

[0066] "Alleviating," "alleviation," and "alleviate" in the context of the disclosed methods all refer to relieving or alleviating the effects or symptoms of a particular disease state, such as PIH.

"Reducing," "reduction," and "reduce" in the context of the disclosed methods all refer to a lessening of the effects or symptoms of a particular disease state, such as PIH.

"Treating," "treatment," and "treat" in the context of the disclosed methods all refer to techniques or methods intended to ameliorate the symptoms or reduce or halt the occurrence of a particular disease state, such as PIH.

"Impurities" include any substance that is undesirable in bakuchiol compositions and that typically results from the isolation of bakuchiol from natural sources. The term impurity includes, but is not limited to, furanocoumarin compounds, including, but not limited to, psoralens, isopsoralens, and other coumarin-type impurities. Impurities also refer to impurities that arise from the synthetic methods used to obtain these compositions.

"Treatment" includes treatment and/or prevention. When used herein, treatment refers to humans as well as other animals.

A "pharmaceutically, cosmetically or therapeutically effective dose or amount" refers to a dosage level sufficient to induce a desired biological or functional result. The result may be alleviation of the signs, symptoms or causes of a disease, skin condition or any other desired alteration of a biological system.

"Placebo" refers to a substitute for the administration of a biologically effective dose or amount of a drug or therapeutic agent sufficient to induce the desired relief of the signs, symptoms, or causes of a disease for which an inactive substance may be used.

A "host," "subject," or "patient" is a living individual, human or animal, to whom the compositions described herein are administered. Thus, the compositions described herein can be used in veterinary as well as human applications, and the term "patient," "subject," or "host" should not be interpreted in a limiting sense. In the case of veterinary applications, the dosage ranges described below can be based on the animal's weight.

As described above, one embodiment of the present disclosure relates to the use of a composition comprising bakuchiol that is substantially free of furanocoumarin impurities for preventing, alleviating, reducing, or treating hyperpigmentation caused by a disease state originating from an inflammatory skin condition. For example, the disclosed methods are suitable for treating post-inflammatory hyperpigmentation (PIH). In some embodiments, PIH may originate from acne. The disclosed methods have demonstrated clinical efficacy in humans in preventing, alleviating, reducing, and treating post-inflammatory hyperpigmentation from skin conditions such as acne, atopic dermatitis, allergic contact dermatitis, incontinentia pigmenti, lichen planus, lupus erythematosus, morphea; and post-inflammatory hyperpigmentation caused by mechanical trauma, ionizing and non-ionizing radiation, burns, lasers, and drug treatments, as well as skin infections using synthetic bakuchiol or furanocoumarin-free psoralea extract bakuchiol compositions. These and other aspects of the present disclosure and various embodiments will become apparent upon reference to the following description.

A. Bakuchiol Compositions

In one embodiment, the present disclosure provides a composition comprising bakuchiol, the bakuchiol being substantially free of impurities, particularly furanocoumarin impurities. The composition is also referred to herein as Bakutrol ^™ . In some embodiments, as shown in document (Hongli Chen and Yuanchao Li, Letters in Organic Chemistry (Organic Chemistry Communications), 2008, 5,467-469), the composition is obtained by organic synthesis from simple compounds or from plants. In some embodiments, the bakuchiol composition is isolated from plants. The plant source of bakuchiol includes the family of plants, the family of plants including but not limited to leguminous family (Luguminosae), Fabaceae (Papilionaceae), Lauraceae (Lauraceae) and Magnoliaceae (Magnoliaceae), and plant genus, the plant genus including but not limited to Psorlea (Psorlea), Sassafras (Sassafras), Magnolia (Magnolia) and Astractylodes (Astractylodes). For example, the bakuchiol composition can be isolated from Psoralea corylifolia L. (Leguminosae) or Psoralea glandulosa L. (Fabaceae). The composition can be obtained from the whole plant or from one or more individual parts of the plant, including but not limited to seeds, stems, bark, branches, tubers, roots, root bark, young shoots, rhizomes, flowers or other reproductive organs, leaves or other aerial parts, or combinations thereof. Methods for isolating bakuchiol from plants can include solvent extraction, supercritical fluid extraction, distillation, physical pressing, or combinations thereof.

Bakuchiol, whose structure is shown below, is a phenolic compound having one hydroxyl group in the aromatic ring and an unsaturated hydrocarbon chain. Although shown in the trans form in the following structure, the benzyl double bond of bakuchiol may also be cis.

The amount of bakuchiol in the purified plant extract (i.e., weight percent (w/w%)) depends on the extraction method and the degree of purification of the crude extract. In one embodiment, as shown in Table 2, the amount of bakuchiol in the extract is between 13.7% and 29.1%. In other embodiments, the amount of bakuchiol in the extract is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In some embodiments, the amount of bakuchiol in the extract is 100%. In other embodiments, the amount of bakuchiol in the composition is no less than 60%. Examples 6-8 provide examples of extracts containing various amounts of bakuchiol.

Although bakuchiol is a bioactive natural product with great potential for preventing and treating various diseases and disease states, there are many limitations associated with the use of this compound. Some limitations include its low concentration in natural sources and the presence of toxic components that co-exist in bakuchiol sources. The impurities present in bakuchiol compositions vary with the bakuchiol source. For example, psoralens, also known as furanocoumarins, are naturally occurring secondary metabolites in Psoralea genus plants (a bakuchiol source) and are also present in many fruits and vegetables. Examples of furanocoumarins that are often found to co-exist with bakuchiol include psoralens and isopsoralens.

Numerous health risks are associated with the handling, topical application, and ingestion of psoralen-containing plants and synthetic psoralens. Psoralens are known to be phototoxic agents that increase the sensitivity of the skin to ultraviolet radiation and promote skin cancer (Epstein (1999) Med. Surg. 18(4): 274-284). Psoralens have been shown to induce growth inhibition in rats (Diawara et al. (1997) Cancer Lett. 114(1-2): 159-160). The gonadal toxicity of crude extracts from Psoralea plants has been directly linked to disruption of the hypothalamic-pituitary-gonadal axis (Takizawa et al. (2002) J. Toxicological Sciences 27(2): 97-105). Oral administration of psoralen, bergamotolactone (5-methoxypsoralen), and cypermethrin (8-methoxypsoralen) in the diet of female rats reduced birth rate, number of implantation sites, puppies, corpora lutea, full and empty uterine weights, and circulating estrogen levels in a dose-dependent manner (Diawara et al. (1999) J. Biochem. Molecular Toxicology 13(3/4):195-203). Psoralens have also been shown to induce mRNAs for the hepatic enzymes CYP1A1 and UGT1A6, suggesting that enhanced estrogen metabolism by psoralens may explain reproductive toxicity and the observed reduction in ovarian follicle function and ovulation (Diawara et al. (2003 May-June) Pediatr Pathol Mol Med. 22(3):247-58.). Because of furanocoumarin toxicity, it is important to remove psoralens and isopsoralens from bakuchiol compositions intended for use in the treatment of post-inflammatory hyperpigmentation or other disease states.

Psoralen and isopsoralen constitute approximately 0.1%-2% of the dry weight of Psoralea seeds and approximately 1%-20% by weight in solvent or supercritical fluid extracts. Crude extracts from Psoralea plants can be obtained by solvent extraction or supercritical fluid extraction, distillation, physical pressing, or a combination of these extraction methods. Enriched bakuchiol compositions can be obtained by chromatography, solvent fractionation (Indian Patent Publication #00570/KOL/2005), distillation, recrystallization, and other wet chemical and physical methods. Published U.S. Patent Application No. 2006/0251749, incorporated herein by reference in its entirety, discloses solvent extraction followed by hydroxylation to decompose the furanocoumarin ring and obtain an enriched bakuchiol composition substantially free of furanocoumarin impurities (e.g., less than 500 ppm or less than 100 ppm of furanocoumarin impurities). The disclosed method comprises the steps of extracting a compound from a plant source, hydrolyzing the crude extract with an alkaline solution under heating, and purifying by methods including, but not limited to, column chromatography, extraction followed by crystallization, solvent layering, recrystallization, and combinations thereof. Applicants have discovered that compositions of bakuchiol-enriched Psoralea extracts substantially free of furanocoumarin impurities can be used to prevent, alleviate, reduce, or treat hyperpigmentation. For example, the disclosed bakuchiol compositions are effective in preventing, alleviating, reducing, or treating post-inflammatory hyperpigmentation (PIH).

The present disclosure also relates to methods for isolating and purifying crude compositions of bakuchiol and related compounds obtained from natural sources. The methods for isolating and purifying these compositions include extracting the compounds from a plant source, hydrolyzing the crude extract with an alkaline solution, and purifying by methods including, but not limited to, column chromatography, extraction followed by crystallization, solvent layering, recrystallization, and combinations thereof. The crude extract purified in this manner is substantially free of furanocoumarin impurities such as psoralen and isopsoralen. As a result, the potential phototoxicity, local irritation, carcinogenicity, and reproductive toxicity associated with these compounds are substantially eliminated.

In some embodiments, the disclosed compositions comprise less than 500 ppm, less than 250 ppm, less than 100 ppm, or less than 50 ppm of total furanocoumarin impurities. The concentration of furanocoumarin impurities can be determined by any method known to those skilled in the art. For example, in one embodiment, the furanocoumarin content can be determined by HPLC.

As described in Example 2, the efficacy of bakuchiol extraction from plant sources was evaluated using six different organic solvent systems under two sets of extraction conditions. The results are set forth in Table 2. Referring to Table 2, it can be seen that bakuchiol can be extracted from Psoralea plants using a number of organic solvents and/or combinations thereof. The amount of bakuchiol in each extract ranged from 13.7% to 29.1% by weight. Other extraction methods include, but are not limited to, _CO2 supercritical fluid extraction and hydrodistillation. Pressed exudates from fresh plant parts, such as seeds, can also be used to obtain bakuchiol compositions from natural sources.

The effectiveness of column chromatography for purifying crude bakuchiol extracts is demonstrated in Example 3 and Table 3. Eight different resin types were specifically evaluated for their ability to separate bakuchiol from furanocoumarin impurities. Both silica gel resin and CG-161 resin demonstrated satisfactory separation. However, column chromatography of crude plant extracts is generally not economically feasible on an industrial scale because it requires expensive equipment and reagents, as well as experienced personnel. The very low loading capacity of these samples, due to the complexity of crude plant extracts, also makes industrial-scale column chromatography difficult.

Example 4 describes an economical method for separating bakuchiol from furanocoumarin impurities. The method involves treating a composition containing furanocoumarin impurities with a base. As illustrated by Scheme 1 below, using NaOH for illustration, base heating is used to open the lactone rings of the furanocoumarins, thereby converting them to the corresponding carboxylate salts. These salts can then be easily separated from the remainder of the mixture by a variety of methods. The disclosed method allows for the preparation of bakuchiol compositions that are substantially free of furanocoumarin impurities (e.g., less than 500 ppm). Such highly pure bakuchiol compositions cannot be obtained using standard chromatographic techniques without the disclosed hydrolysis.

Reaction Scheme 1. Hydrolysis of Furocoumarins

The alkaline solution can include any base capable of opening the lactone ring, including but not limited to sodium hydroxide, potassium hydroxide, calcium hydroxide, lithium hydroxide, or combinations thereof. The solution can have varying concentrations and pH values to maximize conversion to the acid salt. The reaction mixture can also be heated at varying temperatures and pressures to maximize reaction rate, efficiency, and yield.

The reaction process can be followed by HPLC to ensure that the furanocoumarins are fully converted to their corresponding carboxylates. HPLC chromatograms of the crude composition before and after hydrolysis are illustrated in Figure 2. After completion of the reaction (as determined by HPLC), the reaction solution can be treated using a variety of methods, including but not limited to column chromatography, crystallization, solvent layering, precipitation, solvent washing, or a combination thereof. Organic solvents that can be used for solvent layering include but are not limited to petroleum ether, ethyl acetate, ether, hexane, chloroform, propanol, butanol, and dichloromethane, as well as other water-immiscible organic solvents.

The crude extract purified in this manner is substantially free of furanocoumarin impurities such as psoralen and isopsoralen. For example, the purified extract may contain less than 500 ppm, less than 250 ppm, less than 100 ppm, or even less than 50 ppm of furanocoumarin impurities. Furthermore, these highly purified furanocoumarin-free bakuchiol compositions are light brown or red in color and are very stable with respect to the color and composition of the active agent, making them particularly suitable for formulation, storage, and cosmetic applications.

Also included in the present disclosure are methods for analyzing compositions of bakuchiol that are capable of detecting and quantifying impurities. In this embodiment, the method for analyzing a composition of bakuchiol includes the step of analyzing the composition by high pressure liquid chromatography (HPLC). HPLC analysis enables quantification of individual components in a mixture and also provides methods for tracking bakuchiol, psoralens, isopsoralens, and other natural components in Psoralea plants to guide extraction, hydrolysis, and purification methods. A method for analyzing a composition of bakuchiol using high pressure liquid chromatography (HPLC) is described in Example 1 (Table 1).

B. Treatment of Hyperpigmentation Using Bakuchiol Compositions

One embodiment of the present disclosure relates to the use of a composition comprising bakuchiol that is substantially free of furanocoumarin impurities for preventing, alleviating, reducing, or treating hyperpigmentation caused by a condition resulting from an inflammatory skin condition. For example, the disclosed methods include preventing, alleviating, reducing, or treating post-inflammatory hyperpigmentation (PIH). In some embodiments, PIH may result from acne. The disclosure includes formulating the bakuchiol composition in typical cosmetic vehicles and also in skin creams, gel lotions, and other formulations described in more detail below. As shown in the examples, the applicants have demonstrated unexpected human clinical efficacy of the bakuchiol composition in preventing, alleviating, reducing, or treating post-inflammatory hyperpigmentation (PIH), wherein PIH results from skin conditions such as acne, atopic dermatitis, allergic contact dermatitis, incontinentia pigmenti, lichen planus, lupus erythematosus, morphea; and post-inflammatory hyperpigmentation caused by mechanical trauma, ionizing and non-ionizing radiation, burns, lasers, and drug treatments, as well as skin infections.

The disclosed methods include administering to a mammal (e.g., a human patient) an effective amount of a composition comprising bakuchiol that is substantially free of furanocoumarin impurities. For example, the composition may contain less than 500 ppm of furanocoumarin impurities. The composition may contain from about 0.0001% to about 100% bakuchiol. For example, in some embodiments, the composition contains from about 0.1% to about 2% bakuchiol or from about 0.5% to about 1% bakuchiol. In other examples, the composition contains about 0.5% or about 1.0% bakuchiol. In some embodiments, the mammal is a human, and in other embodiments, the mammal is in need of preventing, alleviating, reducing, or treating hyperpigmentation caused by a disease state originating from an inflammatory skin disorder, for example, the mammal may be in need of treatment for PIH.

The present disclosure demonstrates unexpectedly unique biological properties of synthetic or natural bakuchiol compositions. As shown in Example 5 and Table 4, Bakutrol compositions comprising approximately 57.35% bakuchiol exhibit unexpectedly high antioxidant capacity, particularly against superoxide anions (>69,000 μmole TE/g), with a total ORAC value of >92,000 μmole TE/g against five major active species.

Superoxide is an anion with the chemical ^formula _O2- . Chronic inflammatory disease states such as acne vulgaris can have significantly increased production of superoxide anions from keratinocytes, which are stimulated by Gram-positive anaerobic bacteria such as Propionibacterium acnes (P. acnes) (Grange PA. et al., PLoS Pathogens 2009, 5(7)1-14.). Superoxide is highly biotoxic and is disseminated by the immune system to kill invading microorganisms. In phagocytes, superoxide is produced in large quantities by the enzyme NADPH oxidase for an oxygen-dependent killing mechanism of invading pathogens. Superoxide anions and other reactive oxygen species in inflamed skin can also induce melanogenesis, melanocyte proliferation, and melanocyte apoptosis, which are major pathogenic factors of post-inflammatory hyperpigmentation. Therefore, one embodiment of the present disclosure is a method of alleviating, reducing, or treating hyperpigmentation caused by disease states derived from inflammatory skin disorders by reducing superoxide using a composition comprising bakuchiol that is substantially free of furanocoumarin impurities. In one embodiment, the disease state is PIH. In another embodiment, the disclosure provides a method for reducing melanogen production or melanocyte proliferation or inhibiting melanocyte apoptosis, for example, by reducing superoxide anions. The method comprises administering to a mammal an effective amount of a composition comprising bakuchiol substantially free of furanocoumarin impurities. In some embodiments, the mammal is a human, and in other embodiments, the mammal is in need of reducing melanogen production or melanocyte proliferation or inhibiting melanocyte apoptosis.

As demonstrated in Example 6 and Figure 3, a composition comprising 77.02% bakuchiol, substantially free of impurities, particularly furanocoumarin impurities, demonstrated protective effects against oxidative stress induced by 4-tetrabutylphenol (4-TBP). At two concentrations tested, the bakuchiol composition protected against melanocyte cytotoxicity from reactive oxygen species generated by 4-TBP. While not intending to be bound by theory, the applicants believe that the unexpected clinical benefit of synthetic or natural bakuchiol compositions in reducing, alleviating, preventing, or treating post-inflammatory hyperpigmentation (PIH) stems from their unique and unexpected ability to neutralize reactive oxygen species, particularly superoxide anions, and protect melanocytes from oxidative stress in inflammatory disease states that result in reduced epidermal melanoma and/or dermal melanosis.

In addition to its unexpectedly high antioxidant capacity, the applicants discovered that the disclosed bakuchiol composition is not a tyrosinase inhibitor. This is in contrast to other reports disclosing bakuchiol as a skin whitening agent via tyrosinase inhibition (Japanese Patent No. P1107123). This unexpected discovery led the applicants to the presently disclosed method for treating PIH, in which pigmentation occurs in the deep skin layers and tyrosinase inhibitors are ineffective. The disclosed bakuchiol composition without tyrosinase inhibition is shown in Example 7 and FIG4 . Both pure bakuchiol (100%) and enriched bakuchiol with no more than 100 ppm of furanocoumarins from natural sources (77.02%) showed no tyrosinase inhibitory function at eight different doses.

The safety of compositions containing bakuchiol at concentrations of 86.54% and 77.02% bakuchiol was evaluated. As shown in Example 9 and Table 6, based on in vitro and human clinical trials, the Bakutrol (UP256) composition exhibited no eye irritation, no skin irritation to normal or abraded skin, no skin contact sensitization, no phototoxicity, and no mutagenic toxicity. Topical creams of the bakuchiol composition were well tolerated in all human and in vitro studies.

As demonstrated in Example 10, a natural bakuchiol composition (Bakutrol™), extracted and enriched from the seeds of Psoralea corylifolia, containing 77.02% bakuchiol and less than 100 ppm furanocoumarins, was tested in a human clinical trial on subjects with post-inflammatory hyperpigmentation ( ^PIH ) from mild or moderate acne vulgaris. The bakuchiol composition was formulated at 0.5% bakuchiol for topical application. Following daily topical application of 0.5% Bakutrol cream, a significant reduction in post-inflammatory hyperpigmentation (PIH) was observed in all five subjects. As shown in Figure 5, all five subjects experienced a reduction in at least one grade level of PIH severity. Improvement of greater than 50% of the facial area affected by PIH was achieved after 8 weeks of continuous topical application of 0.5% Bakutrol cream (Figure 6). The mean percentage and absolute grade level improvements in both PIH and its severity are summarized in Figures 7 and 8. Greater than 40% improvement in both PIH and severity, or a reduction of greater than one grade level, was achieved as early as four weeks after using the bakuchiol composition. As shown in Figure 9, a substantial reduction in PIH on affected facial skin areas is evident in the photographs of both individuals. Following topical application of Bakutrol cream, both individuals demonstrated a gradual improvement in post-inflammatory hyperpigmentation (PIH) associated with mild and moderate acne.

Table 7 (Example 10) summarizes the clinical outcomes of using a furanocoumarin-free bakuchiol composition (i.e., Bakutrol) compared to popular acne treatment products containing antimicrobial or anti-inflammatory agents, or a combination thereof. The data in Table 7 clearly demonstrate that the furanocoumarin-free bakuchiol composition not only improved inflammatory and non-inflammatory lesion counts, but also significantly improved post-inflammatory hyperpigmentation of the skin. The benefit of the bakuchiol composition on PIH was unexpected given its lack of tyrosinase inhibitory activity.

Table 8 (Example 11) provides data demonstrating a reduction in PIH grade (i.e., degree of pigmentation). The data clearly demonstrate that bakuchiol is more effective than placebo and salicylic acid for treating PIH. Furthermore, the applicants have discovered that bakuchiol (or a composition comprising it) is effective for treating inflammatory lesions, such as acne lesions. Table 9 (Example 11) demonstrates the effectiveness of bakuchiol for treating inflammatory lesions, compared to treatment with a placebo or salicylic acid.

In addition to methods comprising treatment with a composition comprising bakuchiol, the present invention includes embodiments in which a mammal is treated with a composition comprising bakuchiol and salicylic acid. For example, the applicants have discovered that salicylic acid is effective for treating non-inflammatory lesions, while bakuchiol is effective for treating inflammatory lesions. Thus, one embodiment of the present invention relates to a method for treating inflammatory lesions (e.g., acne lesions) comprising administering to a mammal an effective amount of a composition comprising bakuchiol or a pharmaceutically acceptable salt thereof. Another embodiment relates to a method for treating inflammatory and/or non-inflammatory lesions (e.g., acne lesions) comprising administering to a mammal an effective amount of a composition comprising bakuchiol and salicylic acid (or a pharmaceutically acceptable salt thereof). Other embodiments include treating non-inflammatory lesions by administering to a mammal an effective amount of a composition comprising salicylic acid or a pharmaceutically acceptable salt thereof. In some of the foregoing embodiments, the mammal is a human. In other embodiments, the mammal is in need of treatment for inflammatory and/or non-inflammatory lesions such as acne.

In addition to treating pathological conditions, the combination of bakuchiol and salicylic acid is effective for treating any of the aforementioned disease states (e.g., PIH, reducing melanogenesis, reducing melanocyte proliferation, or preventing melanocyte apoptosis, etc.). Accordingly, some embodiments relate to treatments using compositions comprising bakuchiol and salicylic acid. Other embodiments include compositions comprising bakuchiol or a pharmaceutically acceptable salt thereof, salicylic acid or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

The foregoing methods are effective in substantially eliminating inflammatory and/or non-inflammatory lesions. For example, in some embodiments, the methods reduce lesions by about 1% to about 99% or about 10% to about 90%. In other embodiments, the methods reduce lesions by greater than 50%.

The ratio of bakuchiol to salicylic acid is not particularly limited and can be determined by one of ordinary skill in the art based on the desired results. For example, in some embodiments, the weight ratio of bakuchiol to salicylic acid is from about 1:100 to about 100:1. In other embodiments, the weight ratio is from about 10:90, about 20:80, about 30:70, about 40:60, about 50:50, about 60:40, about 70:30, about 80:20 to about 10:90. The composition can be formulated according to any of the formulations described herein.

C. Formulation of Bakuchiol Compositions

The bakuchiol compositions of the present disclosure can be formulated by any method known to those skilled in the art. As shown in Example 8 and Table 5, the compositions of the present disclosure can be formulated in the form of pharmaceutical, cosmetic, or dermatological compositions and can include other components, such as pharmaceutically and/or cosmetically acceptable active ingredients, excipients, adjuvants, carriers, or combinations thereof. Excipients are inert substances that serve as diluents or vehicles for dermatologically and cosmetically acceptable prodrugs and drugs. Examples of such excipients include, but are not limited to, water, buffers, saline, glycerol, hydrated silicon dioxide, propylene glycol, aluminum oxide, carrageenan, cellulose carboxymethyl ether, titanium dioxide, Ringer's solution, glucose solution, mannitol, Hank's solution, preservatives, and other aqueous physiologically balanced salt solutions. Non-aqueous vehicles such as fixed oils, sesame oil, ethyl oleate, or triglycerides can also be used. Other useful formulations include suspending agents containing viscosity-increasing agents such as sodium carboxymethyl cellulose, sorbitol, or dextran.

In Example 8, the composition of the present disclosure is formulated in carbitol, or caprylic triglyceride, or polysorbate-20, or purified water, or a combination of two or more of the above vehicles. The excipients may also contain trace amounts of additives such as EDTA, disodium DDTA, BHA, BHT, diammonium citrate, nordihydroguaiaretic acid, propyl gallate, sodium gluconate, sodium metabisulfite, tert-butylhydroquinone, SnCl ₂ , H ₂ O ₂ and 2,4,5-trihydroxybutyrophenone, vitamin C, vitamin E, vitamin E acetate, phenonip, and other substances that improve isotonicity and chemical stability.

Examples of substances used to adjust the pH of the formulation include sodium hydroxide, sodium carbonate, sodium bicarbonate, pentasodium triphosphate, tetrasodium pyrophosphate, sodium lauryl sulfate, calcium peroxide, phosphate buffer, bicarbonate buffer, tris buffer, histidine, citrate, and glycine, or mixtures thereof. Examples of flavoring agents include, but are not limited to, thimerosal, m-cresol or o-cresol, formalin, fruit extracts, and benzyl alcohol. Standard formulations can be liquid or solid and can be dissolved in a suitable liquid, such as a suspension or solution, for administration. Thus, in non-liquid formulations, excipients can include glucose, human serum albumin, preservatives, and the like, to which sterile water or saline can be added prior to administration.

In one embodiment, the bakuchiol composition can be formulated with other active compounds that target different mechanisms of action for reducing skin pigmentation. Such actives include, but are not limited to, hydroquinone, monobenzyl ethers, arbutin, deoxyarbutin, p-methoxyphenol, N-acetyl-4-S-cysteaminophenol, kojic acid, azelaic acid, glycolic acid, gentisic acid, flavonoids, aloesin, stilbene and stilbene derivatives, licorice extract, bearberry extract, mulberry extract, aloe vera gel, glabridin, vitamin C derivatives, magnesium ascorbyl phosphate, tetrahexyldecyl ascorbic acid, vitamin E derivatives, tranexamic acid and its derivatives, biomimetic compounds of TGF-B proteins, cyanidins, niacinamide, PAR-2 inhibitors, lectins, mimetic glycoproteins, resorcinol and its derivatives, and Nivitol ^™ .

In another embodiment, the composition comprises an anti-inflammatory and antimicrobial agent that can act synergistically with the bakuchiol composition to reduce infection, infection-related inflammation, and accelerate epidermal renewal. Such actives include, but are not limited to, alpha-hydroxy acids, salicylic acid, linoleic acid, tretinoin, benzoyl peroxide, sodium sulfacetyl, clindamycin, erythromycin, dapsone, tetracycline, deoxycycline, minocycline, zinc, estrogens and their derivatives, antiandrogens, sulfur, steroids, cortisone, tazarotene, curcumin extract, gum arabic extract, scutellaria baicalensis extract, green tea extract, and grape seed extract.

In some embodiments, the composition comprises an adjuvant or carrier. Generally, an adjuvant is a substance that generally enhances the biological response of a mammal to a particular biologically active agent. Suitable adjuvants include, but are not limited to, Freund's reagent; other bacterial cell wall components; salts of aluminum, calcium, copper, iron, zinc, magnesium, and tin; silicon dioxide; microdermabrasion agents, polynucleotides; toxoids; serum proteins; viral capsid proteins; other bacterial-derived agents; gamma interferon; block copolymer adjuvants such as Hunter's Titermax adjuvant (Vaxcel.TM., Inc. Norcross, Ga.); Ribi adjuvant (available from Ribi ImmunoChem Research, Inc., Hamilton, Mont.); and saponins and their derivatives, such as Quil A (available from Superfos Biosector A/S, Denmark). Carriers are typically compounds that increase the half-life of the therapeutic composition in the treated recipient. Suitable carriers include, but are not limited to, polymer controlled-release formulations, biodegradable implants, liposomes, nanocapsules, nanoparticles, bacteria, viruses, oils, esters, and glycols.

In other examples, the composition is prepared in the form of a controlled-release formulation that slowly releases the composition to a recipient. As used herein, a controlled-release formulation comprises a bakuchiol composition in a controlled-release vehicle. Suitable controlled-release vehicles are known to those skilled in the art. Examples of controlled-release formulations are biodegradable (i.e., biodigestible) and include capsules.

In one embodiment, a suitable ointment is composed of an effective, non-toxic amount of UP256 (bakuchiol), 65% to 100% (e.g., 75% to 96%) of white soft paraffin, 0% to 15% of liquid paraffin, and 0% to 7% (e.g., 3% to 7%) of lanolin or its derivatives or synthetic equivalents, at the desired concentrations, based on the total weight of the topical formulation. In another embodiment, the ointment may comprise a polyethylene-liquid paraffin base.

In one embodiment, a suitable cream consists of an emulsifying system together with a desired concentration of UP256 (Bakuchiol) synthesized and/or isolated from the plant or plants provided above. The emulsifying system preferably comprises 2% to 10% of polyoxyethylene alcohol (for example, a mixture available under the trademark Cetomacrogol™ 1000), 10% to 25% of stearyl alcohol, 20% to 60% of liquid paraffin and 10% to 65% of water; together with one or more preservatives, for example 0.1% to 1% of N,N″-methylenebis[N′-[3-(hydroxymethyl)-2,5-dioxo-4-imidazolidinyl]urea] (available under the name Imidacloprid USNF), 0.1% to 1% of an alkyl 4-hydroxybenzoate (for example, a mixture available under the trademark Nipastat from Nipa Laboratories), 0.01% to 0.1% of sodium 4-hydroxybenzoate butyrate (available under the trademark Nipabutylsodium from Nipa Laboratories) and 0.1% to 2% of phenoxyethanol.

In one embodiment, a suitable gel consists of a semisolid system in which a liquid phase is confined within a three-dimensional polymer matrix with a high degree of crosslinking. The liquid phase may comprise water together with the desired amount of UP256 (bakuchiol), 0.01% to 20% of a water-soluble additive such as glycerol, polyethylene glycol, or propylene glycol, and 0.01% to 10%, preferably 0.5% to 2%, of a thickener, which may be a natural product such as tragacanth, pectin, carrageenan, agar, and alginic acid, or a synthetic or semisynthetic compound such as methylcellulose and carbopol (carbopol); together with one or more preservatives, such as 0.1% to 2% of methyl 4-hydroxybenzoate (methylparaben) or phenoxyethanol-differential. Another suitable formulation consists of the desired amount of UP256 (bakuchiol), together with 70% to 90% polyethylene glycol (e.g., a polyethylene glycol ointment containing 40% polyethylene glycol 3350 and 60% polyethylene glycol 400, prepared according to the United States Formulary (USNF)), 5% to 20% water, 0.02% to 0.25% of an antioxidant (e.g., butylated hydroxytoluene), and 0.005% to 0.1% of a chelating agent (e.g., ethylenediaminetetraacetic acid (EDTA)).

The term soft paraffin as used above includes white and yellow soft paraffin, which are cream or ointment-like. The term lanolin includes both natural wool fat and refined wool fat. Lanolin derivatives particularly include lanolin that has been chemically modified to alter its physical or chemical properties, and synthetic equivalents of lanolin particularly include synthetic or semi-synthetic compounds and mixtures known and used in the pharmaceutical and cosmetic fields as lanolin substitutes and may be referred to, for example, as lanolin substitutes.

A suitable synthetic equivalent of lanolin that may be used is the material available under the trade name Softisan™, known as Softisan 649. Softisan 649, available from Dynamit Nobel Aktiengesellschaft, is a glycerol ester of natural vegetable fatty acids, isostearic acid and fatty acids; its properties are discussed by H. Hermsdorf in Fette, Seifen, Anstrichmittel, Issue 84, No. 3 (1982), pp. 3-6.

The other substances mentioned above as components of suitable ointment or cream bases and their properties are discussed in standard reference texts, such as the United States Pharmacopoeia. Cetomacrogol 1000 has the general formula _CH3 ( _CH2 ) _m ( _OCH2CH2 ) _nOH , where m can be 15 or ₁₇ and n can be 20 to 24. Butylated hydroxytoluene is 2,6-di-tert-butyl-p-cresol. Nipastat is a mixture of methyl, ethyl, propyl, and butyl 4-hydroxybenzoate.

The compositions disclosed herein can be prepared by conventional pharmaceutical techniques. Thus, for example, the compositions can be conveniently prepared by mixing together soft paraffin, liquid paraffin, if present, and lanolin or a derivative or synthetic equivalent thereof at an elevated temperature, for example, 60° C. to 70° C. The mixture can then be cooled to room temperature and, after addition of the hydrated crystalline calcium salt of mupirocin, along with the corticosteroid and any other ingredients, stirred to ensure adequate dispersion.

Finally, bakuchiol has a partition coefficient of log P = 6.13. The partition coefficient of a chemical compound provides a thermodynamic measure of its hydrophilicity/lipophilicity balance, and therefore its potential bioavailability. Having a partition coefficient of 6.13 indicates that the compound has high cell membrane penetration and bioavailability when formulated in a delivery system. Skin penetration of bakuchiol, the active compound in a skin cream, was quantified in an in vitro assay on isolated human skin. The results showed good skin penetration and bioavailability. In some embodiments, the disclosed compositions include a skin penetration enhancer.

D. Administration of Bakuchiol Compositions

The compositions of the present disclosure can be administered by any method known to those of ordinary skill in the art. For example, the disclosed compositions can be administered orally or topically. Methods of administration include, but are not limited to, enteral (oral) administration, parenteral (intravenous, subcutaneous, and intramuscular) administration, and topical application. In some embodiments, the compositions are administered topically.

The bakuchiol composition can be present in a final skin care product for PIH at a level of 0.001% to 99.9% by weight. In some embodiments, the composition comprises 0.1% to 2% bakuchiol. In other embodiments, the composition comprises 0.5% or 1.0% bakuchiol. In some embodiments, the amount of the bakuchiol composition in a PIH skin cream is 0.5%-1%. The disclosed methods comprise orally or topically administering to a mammal a therapeutically effective amount of a composition comprising bakuchiol that is fully synthesized or isolated from a natural source (or a combination thereof) and is substantially free of impurities, particularly furanocoumarin impurities (e.g., less than 500 ppm).

The therapeutic agents of the present disclosure can be administered topically for topical administration of therapeutic compositions by any suitable method known to those skilled in the art. Such methods of administration include, but are not limited to, ointment, gel, lotion, or cream bases or in emulsion form, in plaster form, dressings or masks, non-stick gauze, bandages, cotton swabs, or wipes. Such topical application can be administered topically to any affected area using any standard method known for topical administration. The therapeutic composition can be administered in a variety of unit dosage forms depending on the method of administration. For a particular mode of delivery, the therapeutic composition can be formulated with the excipients described above. The therapeutic compositions of the present disclosure can be administered to any recipient, preferably to a mammal, and more preferably to a human. The particular mode of administration will depend on the disease state being treated.

Regardless of the route of administration, specific dosages are calculated based on the approximate body weight of the recipient. Further optimization of the calculations necessary to determine appropriate dosages for treatment with each of the above-described formulations is routinely performed by one of ordinary skill in the art and is within the scope of their routine work without undue experimentation, particularly in light of the dosage information and assays disclosed herein. These dosages can be determined using established assays for use in conjunction with appropriate dose-response data. In some embodiments, the dosage of the composition comprising bakuchiol is 0.001 mg to 200 mg per kilogram of body weight.

The following examples are offered for purposes of illustration and not limitation.

Example

Example 1

Quantification of bakuchiol, psoralen, and isopsoralen by HPLC

The amounts of bakuchiol, psoralen, and isopsoralen in extracts, fractions, process raw materials, ingredients, and final formulated products were quantified by high-pressure liquid chromatography (HPLC) using a photodiode array detector (HPLC/PDA). The target compounds were eluted from a Luna phenyl-hexyl column (250 mm x 4.6 mm) using a gradient of 36% acetonitrile (ACN) or methanol and water to 100% ACN in 12 minutes, followed by 100% ACN in 3 minutes. The detailed HPLC conditions used are described in Table 1. The chromatogram of the HPLC separation is shown in Figure 1. Commercially available pure bakuchiol, psoralen, and isopsoralen were used as quantitative standards, and the target compounds were identified and quantified based on retention time and UV peak area. The retention times of bakuchiol, psoralen, and isopsoralen were 18.19 minutes, 7.33 minutes, and 7.95 minutes, respectively.

Table 1. HPLC conditions for quantification of bakuchiol, psoralen, and isopsoralen

Example 2

General method for extracting bakuchiol from Psoralea plants

Method A - Solvent (100 mL) and Psoralea corylifolia seed powder (10 g) were added to a flask and the mixture was shaken on a manual shaker at room temperature for 1 hour. The mixture was then passed through a filter and the filtrate was collected. The extraction process was repeated using fresh solvent, the combined filtrates were removed from the solvent on a rotary evaporator, and the residue was dried under high vacuum.

Method B - Solvent (50 mL) and Psoralea corylifolia seed powder (10 g) were added to a flask and the mixture was refluxed for 40 min. The solution was then filtered and the extraction process was repeated twice using fresh solvent. The filtrates were combined and the solvent was evaporated to obtain a dry extract.

Following the extraction method described above, sample plant matter was extracted using the following solvents: dichloromethane (DCM), ethyl acetate (EtOAc), acetone, methanol (MeOH), petroleum ether (BP 35-60°C), and petroleum ether (BP 60-90°C). The extracts and plant matter were then analyzed by HPLC analysis as described in Example 1. The results are set forth in Table 2.

Table 2. Quantification of various Psoralea corylifolia extracts

Example 3

Chromatographic method for purification of bakuchiol extract

Various chromatographic methods were used to purify bakuchiol from a crude solvent extract isolated from the seeds of Psoralea corylifolia using the method described in Example 2. The efficiency of a specific column enrichment method was demonstrated as a method for obtaining high-purity bakuchiol free of furanocoumarin contaminants, particularly psoralen/isopsoralen contaminants. Briefly, individual empty column shells (1.3 cm inner diameter (ID) and 20 mL volume, obtained from Bio-Rad) were filled with different media and eluted using different solvents in an attempt to separate the furanocoumarin impurities from bakuchiol. Fractions (10 mL per fraction) were collected in test tubes and analyzed using silica gel TLC plates developed with 20% EtOAc/petroleum ether. The target compounds, bakuchiol, psoralen, and isopsoralen, were identified based on their retention times determined using solutions of standard compounds. The results are summarized in Table 3. Many of the methods described in Table 3 are used to isolate furanocoumarins and bakuchiol from synthetic and natural sources, however the cost of the methods may not be economically feasible for large-scale production.

Table 3. Summary of separation of bakuchiol from furanocoumarins in crude extracts of Psoralea corylifolia by column chromatography

Example 4

Hydrolysis of extracts isolated from seeds of Psoralea corylifolia

A hexane extract or a _CO supercritical fluid extract of Psoralea corylifolia seeds containing approximately 25% bakuchiol is mixed with a 1M NaOH solution. The solution is heated to 80°C or higher in a reaction vessel for at least 1 hour. A small portion of the solution is periodically taken out from the flask and analyzed by HPLC as described in Example 1. The reaction is stopped and the peaks showing psoralen and isopsoralen disappear completely after HPLC analysis. The reaction mixture is then cooled to room temperature and the aqueous phase is removed. After the solution is washed multiple times with a saturated NaCl solution, the organic layer is extracted with ethyl acetate or other organic solvents. The organic solution is filtered, washed, dried, and evaporated to produce a brownish-red syrup having a bakuchiol content of not less than 50% and a total of not more than 100ppm of psoralen and isopsoralen (isopsoralen).

Example 5

Antioxidant properties of bakuchiol compositions without furanocoumarins

A natural bakuchiol composition (Batch No. UP256-0906MP) containing 57.35% bakuchiol and less than 100 ppm of psoralen and isopsoralen (isopsoralen) in total was evaluated for its antioxidant capacity against peroxyl radicals, hydroxyl radicals, peroxynitrites, superoxide anions, and singlet oxygen at Brunswick Laboratories in Norton, MA, USA. The total oxygen radical absorbance capacity of the bakuchiol composition was determined according to published techniques (Ou, B. et al., J Agric and Food Chem, 2001, 49(10):4619-4626; Prior, RL. et al., J Agric and Food Chem, 2005, 53:4290-4302). The results are tabulated in Table 4.

Table 4: Antioxidant properties of Bakutrol ^™ (UP256)

Example 6

Evaluation of the antioxidant protective effect of bakuchiol compositions against 4-TBP cytotoxicity

The antioxidant properties of a natural bakuchiol composition containing 77.02% bakuchiol and less than 100 ppm of psoralen and isopsoralen (isopsoralen) combined were tested by evaluating its ability to prevent oxidative stress induced by 4-tert-butylphenol (4-TBP) during a 5-day treatment period with the compound at a concentration at the 95% action dose. Oxidative stress was measured by examining the production of reactive oxygen species (ROS) using the Image-iT Live Green Reactive Oxygen Species Detection Kit (InVitrogen). In this assay, carboxy-2',7'-dichlorodihydrofluorescein diacetate was added to cultured cells for 30 minutes, where it diffused into melanocytes and underwent intracellular ester hydrolysis to 2',7'-dichlorofluorescein (DCF), which reacted with ROS to produce fluorescent DCF. After 5 days of treatment with 200 μM or 400 μM 4-TBP, ROS production in melanocytes demonstrated a dose response (i.e., mild to strong, respectively), whereas untreated and DMSO-treated melanocytes showed no ROS production. When the test compound was included in the treatment regimen, UP256 (bakuchiol) exhibited strong antioxidant properties as shown in Figure 3.

Example 7

Tyrosinase inhibitory activity of bakuchiol compositions

Two natural bakuchiol compositions containing 77.02% bakuchiol (100% purity) were tested for tyrosinase inhibitory activity. Both materials contained less than 100 ppm total psoralens and isopsoralens (isopsoralens).

Tyrosinase inhibition assays were performed using the method reported by Jones et al. (2002) Pigment. Cell Res. 15 :335. Using this method, the conversion of L-DOPA, the tyrosinase substrate, to dopachrome was tracked by monitoring absorbance at 450 nm. Tyrosinase was prepared in 50 mM potassium phosphate buffer at pH 6.8 (assay buffer) at 2000 U/ml and stored in 1 ml aliquots at -20°C prior to use. For assay use, the stored enzyme solution was thawed and diluted to 200 U/ml with assay buffer. A 2 mM substrate treatment solution, L-DOPA, was prepared in assay buffer for each assay. Samples were dissolved in 10% DMSO (0.5 ml) and diluted to 5 ml with assay buffer. The reaction mixture contained 0.050 ml of 2 mM L-DOPA, 0.050 ml of 200 U/ml mushroom tyrosinase, and 0.050 ml of inhibitor. The reaction volume was adjusted to 200 μl with assay buffer. In 96-well Falcon 3097 flat-bottom microtiter plates (Beckton Dickinson, NJ), the assay was performed. A WALLAC 1420 multi-label counter (Turku, Finland) was used to detect the appearance of dopachrome. The average rate was determined from the linear enzyme rate as measured by the change in absorbance (Δ A ₄₅₀ ) at 450 nm per minute. The percentage inhibition of tyrosinase by the test sample was determined using formula (1) by comparing the absorbance of the sample with that of the control:

(Negative control absorption - sample absorption)/negative control absorption × 100 (1)

As shown in FIG4 , the two bakuchiol compositions exhibited no tyrosinase inhibitory activity, while the positive control (kojic acid) showed dose-responsive tyrosinase inhibition with an IC ₅₀ value of 63.9 μM.

Example 8

Formulation of bakuchiol compositions in the form of cosmetic creams, gels and lotions

As shown below, two natural bakuchiol compositions containing 86.54% bakuchiol and 77.02% bakuchiol were formulated in the form of a cosmetic vehicle or a complex skin cream, gel or lotion.

Preparation A

Preparation B

Preparation C

Table 5. Formulation D

Example 9

Evaluation of the safety attributes of bakuchiol compositions

Two natural bakuchiol compositions containing 86.54% bakuchiol and 77.02% bakuchiol, with less than 100 ppm of psoralen and isopsoralen (isopsoralen) combined, were formulated as either cosmetic vehicles or complex skin creams and tested for their safety profiles in in vitro models or in human clinical trials. As shown in Table 6, the bakuchiol compositions exhibited no eye irritation, no skin irritation, no skin allergy contact sensitization, and no phototoxicity. The compositions exhibited a solid safety profile (20% to 100% bakuchiol by weight) and good skin penetration properties over a wide range of concentration levels.

Table 6: Results of Bakutrol ^™ Safety Trials

Example 10

Clinical evaluation of a furanocoumarin-free bakuchiol composition

A natural bakuchiol composition (Bakutrol ^™ ), extracted and enriched from the seeds of Psoralea corylifolia and containing 77.02% bakuchiol and less than 100 ppm furanocoumarins, was formulated in a cosmetic skin cream (Formulation D, Example 8) and tested in a human clinical trial. The study was a pilot, open-label, human study to evaluate the clinical efficacy of Bakutrol ^™ at a 0.5% concentration following topical application. Five subjects who met the inclusion/exclusion criteria were included in the study to evaluate the efficacy of the natural bakuchiol composition in improving post-inflammatory hyperpigmentation (PIH). The study duration was 12 weeks. Subjects were instructed to apply Bakutrol ^™ 0.5% cream twice daily, morning and evening, and returned to the site for a total of nine visits, including the screening visit. Evaluations included an Investigator Global Assessment of Skin Condition (IGA), the overall grade and severity of post-inflammatory hyperpigmentation (PIH) and other relevant skin disease states, including erythema, dryness, flaking, oiliness, and safety and tolerability assessments. Individual questionnaires included safety and compliance questions regarding irritation, skin comfort, and use of other products and sunscreens. Photographs were taken at baseline, week 4, week 8, and week 12. Changes in PIH severity from baseline, PIH grade from baseline, and the percentage of success according to the IGA scale were recorded and analyzed. The following 6 levels of PIH severity (0 = none, 1 = mild, 2 = mild, 3 = moderate, 4 = moderately severe, 5 = severe) and the total area of the buccal surface affected by PIH were used for clinical output analysis.

As shown in Figure 5, all five individuals treated with a topical cream containing Bakutrol ^™ at 0.5% had a reduction in PIH severity of at least one grade. The percentage of PIH-infected facial areas improved by more than 50% after 8 weeks of continued application (see Figure 6). The average percentage and absolute grade-level improvements in both PIH and its severity are summarized in Figures 7 and 8. As early as 4 weeks after using a natural bakuchiol composition extracted and enriched from the seeds of Psoralea corylifolia and containing 77.02% bakuchiol and less than 100 ppm of furanocoumarins, improvements of more than 40% or more than one grade-level in both PIH and severity were achieved. The significant reduction in PIH on the affected facial skin areas is clearly demonstrated in the photographs of two individuals shown in Figure 9. After topical application of Bakutrol ^™ cream, both individuals showed a gradual improvement in post-inflammatory hyperpigmentation (PIH) of the skin associated with mild and moderate acne.

Table 7 summarizes the clinical outcomes of using a furanocoumarin-free bakuchiol composition compared to popular acne treatment products containing antimicrobial or anti-inflammatory agents, or a combination thereof. The data clearly demonstrate that the furanocoumarin-free bakuchiol composition not only improved inflammatory and non-inflammatory lesion counts but also significantly improved post-inflammatory hyperpigmentation of the skin. As demonstrated in Example 7, the PIH benefit derived from the bakuchiol composition was unexpected given its lack of tyrosinase inhibition.

Table 7. Summary of clinical reports on Bakutrol and OTC drugs

Example 11

Evaluation of the effects of a furanocoumarin-free bakuchiol composition on reducing post-inflammatory hyperpigmentation

The safety and efficacy of 0.5% (wt/wt) bakuchiol (UP256) cream for the treatment of acne-related PIH were evaluated in a double-blind, placebo-controlled study. The study evaluated 0.5% bakuchiol cream, 2% salicylic acid cream, and placebo cream (vehicle) in an Asian population. Participants were instructed to apply the study cream to their face twice daily (AM/PM). They were given application instructions and provided sunscreen.

The study subjects consisted of male and female individuals who were older than 18 years and younger than 40 years and in generally good health as determined by medical records. 18 individuals were enrolled in the bakuchiol study group, 20 individuals were enrolled in the salicylic acid (SAL) study group, and 19 individuals were enrolled in the placebo study group.

The investigators and research staff discussed and agreed upon a specific definition of PIH, such as that associated with acne or other tissue damage (this does not include freckles, solar lentigines, or melasma). PIH grade is a measure of the severity of hyperpigmentation (higher numbers = more severe hyperpigmentation). The patient population had a PIH grade > 3 and acne of mild to moderate grade 2-3, with the primary factor being PIH associated with inflammatory current or post-inflammatory acne.

Main research objectives:

1. PIH IGA Timeframe: Baseline and Weeks 2, 4, and 8

2. PIH% Distribution Timeframes: Baseline and Weeks 2, 4, and 8

PIH efficacy was evaluated based on the change from baseline (p < 0.05) and on other treatment groups (p < 0.05) using t-test and/or analysis of variance.

Secondary objective: Safety assessment

Individual assessment questionnaires and tolerance assessments were collected at baseline and at weeks 2, 4, and 8. Urine pregnancy tests were collected at baseline and week 8 for females of childbearing potential.

Data Analysis

Collect the following data:

1. Change in PIH severity from baseline;

2. Change in PIH grade from baseline; and

3. Change in Lesion Count from Baseline

result:

Data were analyzed at each visit based on investigator assessments and from photographs taken at defined time points. Photographs from baseline, week 4, and week 8 were evaluated by a second investigator team and two independent dermatologists before the study was folded to further confirm that the population met criteria. Analyzed data were based on confirmed assessments; week 2 photographs were unavailable, so week 2 data were not included in the analysis. Table 8 summarizes the data.

Table 8. Changes in PIH grades in the study groups

The Bakutrol (UP256) group showed a significant change from baseline in PIH grade (i.e., lower PIH grade) at Week 8 (p < 0.05). Furthermore, the Bakutrol group showed a significant change from placebo at Week 8 (p < 0.05). The data also showed that the Bakutrol-treated group was the only group with a significant p-value for both time and treatment (p = 0.0083).

Table 9. Inflammatory acne lesions in all groups

The Bakutrol (UP256) group showed significant changes from baseline at both Weeks 4 and 8 (p<0.001). The Bakutrol group also showed a significant change over placebo at Week 8 (p<0.05). Furthermore, UP256 was the only group to achieve a greater than 50% reduction in inflammatory lesions at Week 8 (57%).

There were no significant changes in the non-inflammatory lesion category in the Bakutrol (UP256) group (data not shown); however, the salicylic acid-treated group achieved a p-value < 0.05 in the non-inflammatory lesion category at week 4 of treatment.

Table 10: Overview of the questionnaire and investigator safety assessment

in conclusion:

Results showed that Bakutrol (UP256) cream significantly reduced post-inflammatory hyperpigmentation (PIH) associated with acne after only 4 weeks of topical application. UP256 also significantly reduced inflammatory acne lesions (<0.05) by 57% after 8 weeks.

Bakutrol (UP256) cream had a good safety profile and was well tolerated by study participants. The cosmetic acceptability of the cream was considered superior to or equal to that of their previous topical over-the-counter (OTC) acne treatments.

Example 12

Treatment of inflammatory and noninflammatory acne lesions

A composition comprising both bakuchiol and salicylic acid was prepared. The composition was used to treat acne patients with inflammatory, non-inflammatory, or both types of lesions. The composition was effective in treating both inflammatory and non-inflammatory lesions, with a p-value < 0.05 at week 4 of treatment. The reduction in lesions was from about 10% to about 90%.

The various embodiments described above can be combined to provide further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent applications mentioned in the specification and/or listed in the application data sheets are incorporated herein by reference in their entireties. If necessary, various aspects of the embodiments can be modified to employ concepts from the various patents, applications, and publications to provide further embodiments. These and other changes can be made to the embodiments in light of the above detailed description. Generally, in the following claims, the terms used should not be construed to limit the rights to the specific embodiments disclosed in the specification and claims, but should be construed to include all possible embodiments, along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

Claims (13)

1. Use of a composition comprising bakuchiol or a pharmaceutically acceptable salt thereof in the preparation of a topical formulation for the prevention, relief, reduction or treatment of hyperpigmentation caused by a disease state originating from an inflammatory skin condition. 2. The use as described in claim 1, wherein the psoralen is a synthetic psoralen. 3. The use as described in claim 2, wherein the synthesized psoralen is a mixture of stereoisomers. 4. The use as described in claim 1, wherein the psoralen is isolated from plant species of the genera *Psoralea*, *Sassafras*, *Magnolia*, or *Atractylodes*. 5. The use as described in claim 1 or 4, wherein the psoralen is isolated from the whole plant or from seeds, stems, bark, branches, roots, root bark, buds, flowers or other reproductive organs, leaves or other above-ground parts or combinations thereof. 6. The use as claimed in claim 5, wherein the stem comprises tubers and rhizomes. 7. The use as claimed in claim 1, wherein the hyperpigmentation originating from inflammatory skin conditions arises from acne, atopic dermatitis, allergic contact dermatitis, pigment incontinence, lichen planus, lupus erythematosus, morphine, mechanical trauma, ionizing or non-ionizing radiation, burns, laser or drug treatment, skin infections, or combinations thereof. 8. The use as claimed in claim 1, wherein the hyperpigmentation caused by a disease state originating from an inflammatory skin condition is acne patches. 9. The use as claimed in claim 1, wherein the composition comprises 0.001% to 99.9% by total weight of psoralen and a pharmaceutically acceptable, dermatologically acceptable, or cosmetically acceptable carrier. 10. The use as claimed in claim 1, wherein the composition further comprises one or a mixture of skin whitening agents selected from hydroquinone, monobenzyl ether, arbutin, deoxyarbutin, p-methoxyphenol, N-acetyl-4-S-cysteine, kojic acid, azelaic acid, glycolic acid, gentian acid, flavonoids, aloe vera extract, stilbene and stilbene derivatives, licorice extract, arbutin extract, mulberry extract, aloe vera gel, glycyrrhizin, vitamin C derivatives, vitamin E derivatives, tranexamic acid and its derivatives, biomimetic TGF-B protein, cyanidin, nicotinamide, PAR-2 inhibitors, lectins, glycoproteins, resorcinol and its derivatives, and Nivitol. 11. The use as claimed in claim 10, wherein the vitamin C derivative comprises magnesium ascorbate phosphate and tetrahexyldecyl ascorbic acid. 12. The use as claimed in claim 1, wherein the composition further comprises one or a mixture of skin modifiers selected from α-hydroxy acids, salicylic acid, linoleic acid, retinoic acid, benzoyl peroxide, sulfamethoxazole sodium, clindamycin, erythromycin, dapsone, tetracycline, deoxytetracycline, minocycline, zinc, estrogens and their derivatives, anti-androgens, sulfur, steroids, cortisone, tazarotene, curcumin extract, gum arabic extract, scutellaria baicalensis extract, green tea extract, and grape seed extract. 13. The use as claimed in claim 1, wherein the composition is formulated in an ointment, gel, lotion, cream base as an emulsion, plaster, dressing or mask, non-adhesive gauze, bandage, cotton swab or wiping cloth.